Microbial transformation process for producing an antihypertensive product

ABSTRACT

Fermentation of the microorganism Streptomyces sp. (MA6750) ATCC No. 55042 in the presence of the Angiotensin II (A II) receptor antagonist of the following structure: ##STR1## yields an analog having a shikimate sugar-like moiety attached to the tetrazole, the said analog which is also an A II antagonist useful in the treatment of hypertension and congestive heart failure and other indications known to respond to A II angatonists.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel compound of the formula 1 and a novelprocess for the preparation of the compound of the formula I: ##STR2##comprising fermentation of the compound of formula II: ##STR3## with themicroorganism Streptomyces sp. (MA6750), ATTC No. 55042. Compounds I andits precursor II are Angiotensin II (A II) receptor antagonists usefulin the treatment of human hypertensive diseases.

Angiotensin II is an octapeptide hormone produced mainly in the bloodduring the cleavage of Angiotensin I by angiotensin converting enzyme(ACE). ACE is localized on the endothelium of blood vessels of lung,kidney and many other organs. A II is the end product of therenin-angiotensin system (RAS), a system that plays a central role inthe regulation of normal blood pressure and seems to be criticallyinvolved in hypertension development and maintenance, as well ascongestive heart failure. A II is a powerful arterial vasconstrictorthat exerts its action by interacting with specific receptors present oncell membranes. A II receptor antagonism is one of the possible modes ofcontrolling the RAS.

2. Brief Description of Disclosures in the Art

Copending U.S. application Ser. No. 516,286, filed May 4, 1990,specifically discloses the substrate compound used in this invention anddesignated herein as compound II.

Copending U.S. application Ser. No. 623,479, filed Nov. 4, 1990,discloses the microbial biotransformation products produced when adifferent A II reception antagonist is employed as a substrate compoundin a fermentation with the same Streptomyces sp. employed in thetransformation in the instant invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an ¹ H nuclear magnetic resonance (NMR) spectrum taken at 400MHz of compound I in acetone d₆.

FIG. 2 is an ¹³ C NMR spectrum taken at 400 MHz of compound I inmethanol d₄.

FIG. 3 is an ¹³ C NMR spectrum taken at 400 MHz of compound II inmethanol d₄.

SUMMARY OF THE INVENTION

A new antihypertensive biotransformation product, compound I wasobtained by the fermentation of the microorganism Streptomyces sp. (MA6750), ATCC No. 55042, in the presence of substrate compound II. Thebiotransformation was accomplished under submerged aerobic conditions inan aqueous carbohydrate medium containing a nitrogen nutrient at a pH ofabout 7 for a sufficient time to produce compound I.

Compound I exhibits A II antagonist activity, i.e., has an inhibitoryconcentration (IC₅₀) of less than 50 μm in an assay that measures theconcentration of potential A II antagonist needed to achieve 50%displacement of the total ¹²⁵ I-Sar¹ Ile⁸ -angiotensin II specificallybound to a membrane preparation derived from rabbit aortae, andtherefore it is antihypertensive agents. This assay is also referred toherein as the "IC₅₀ " assay.

Also provided by this invention is a pharmaceutical compositioncontaining a therapeutically effective amount of compound I incombination with a pharmaceutically acceptable non-toxic carrier orexcipient.

In addition, there is provided a method of treating a human host tocontrol hypertensive disorders or for treating congestive heart failurecomprising administering to the host a therapeutically effective amountof compound I.

DETAILED DESCRIPTION OF THE INVENTION

The novel compound of this invention is compound I: ##STR4##

The novel process of this invention comprises fermentation of themicroorganism Streptomyces sp. in the presence of substrate compound II##STR5## in a nutrient medium, and isolation of the resultingbiotransformation product, compound (I), in a conventional manner. Abiologically pure sample of Streptomyces sp. (MA6750) is currentlyavailable in the permanent culture collection of the American TypeCulture Collection, 12301 Parklawn Drive in Rockville, Md., from whichit is available under the Accession Number ATCC 55042.

The following is a general description of Streptomyces sp. strainMA6750. Observations of growth, general cultural characteristics andcarbon source utilization were made in accordance with the methods ofShirling and Gottleib, Internat. J. System. Bacteriol. 16: 313-340(1966). Chemical composition of the cells was determined using themethods of Lechevalier and Lechevalier in Actinomycete Taxonomy, A.Dietz and D. W. Thayer, Ed., Society for Industrial Microbiology (1980).Coloration of the culture was determined by comparison with colorstandards contained in the Inter-Society Color Council-National Bureauof Standards Centroid Color Charts (U.S. Dept. of Commerce NationalBureau of Standards supplement to NBS Circular 553, 1985).

ANALYSIS OF CELL WALL COMPOSITION

Peptidoglycan contains L-diaminopimelic acid. Whole cell analysis ofcarbohydrates reveals glucose, ribose, rhamnose and xylose (trace).

GENERAL GROWTH CHARACTERISTICS

Good growth on yeast malt extract (w/o trace elements), glycerolasparagine, inorganic salts-starch, oatmeal, Bennet's, and trypticasesoy agars. Growth occurs at 27° and 37° C. Culture also grows well inliquid media such as yeast dextrose and tryptone yeast extract broths.

COLONY MORPHOLOGY (ON YEAST MALT EXTRACT AGAR)

Substrate mycelium is slightly brown (55 s.Br) and colonies are opaque,raised, erose and rubbery. The colony surface is rough textured. Aerialmycelia appear after 4 days incubation and are white (263 White) incolor. Spore mass, when present, is light grayish-yellow brown (79l.gy.yBr)

MICROMORPHOLOGY

Aerial mycelium (0.76 μm dia.) arises from a substrate mycelium and isbranched and flexous. In mature cultures, the aerial mycelium commonlyterminates is spores borne in flexous chains.

MISCELLANEOUS PHYSIOLOGICAL REACTIONS

Culture produces melanoid pigments in tryptone yeast extract broth, H₂ Sin peptone yeast extract/iron broth. Starch is not hydrolyzed. Browndiffusible pigment produced on Bennet's and yeast malt extract/traceelement agars.

YEAST EXTRACT-MALT EXTRACT MEDIUM

Aerial Mycelium and/or Spores: Light grayish yellow brown (l.gy.yBr 79),flexous sporphores

Soluble Pigments: Brown

Reverse Color: Slight brown (s.Br 55).

GLUCOSE-ASPARAGINE MEDIUM

Aerial Mycelium and/or Spores: Yellow-white (92), flexous sporphores

Soluble Pigments: None noted

Reverse Color: Deep orange yellow (d.Oy 72).

INORGANIC SALTS-STARCH MEDIUM

Aerial Mycelium and/or Spores: Light gray (l.Gy 264), flexous sporphores

Soluble Pigments: None noted

Reverse Color: Deep orange yellow (d.Oy 72).

OATMEAL MEDIUM

Aerial Mycelium and/or Spores: Yellow-white (92), flexous sporphores

Soluble Pigments: None noted

Reverse Color: Light orange yellow (l.Oy 70).

    ______________________________________                                        Carbohydrate Utilization Pattern of                                           Streptomyces sp. MA6750 at 21 Days                                            Carbon Source     Utilization*                                                ______________________________________                                        D-arabinose       2                                                           L-arabinose       3                                                           D-fructose        3                                                           inositol          3                                                           α-D-lactose 3                                                           β-D-lactose  3                                                           D-maltose         3                                                           D-mannitol        3                                                           D-raffinose       3                                                           L-rhamnose        3                                                           sucrose           3                                                           D-xylose          3                                                           L-xylose          0                                                           α-D-glucose (control)                                                                     3                                                           ______________________________________                                         *3 = good utilization                                                         2 = moderate utilization                                                      1 = poor utilization                                                          0 = no utilization                                                       

In general, compound (I) can be produced by culturing (fermenting) theabove-described microorganism in the presence of an appropriateconcentration of substrate compound (II) in an aqueous nutrient mediumcontaining sources of assimilable carbon and nitrogen, preferably undersubmerged aerobic conditions (e.g. shaking culture, submerged culture,etc.). An appropriate concentration of the parent compound in theaqueous medium ranges from 0.05 mg/ml to 0.2 mg/ml, preferably 0.05mg/ml; less than 0.05 mg/ml is inefficient and greater than 0.2 mg/mlcan inhibit the culture. The aqueous medium is incubated at atemperature between 26° C. and 29° C., preferably 27° C.; culture growthwill be inhibited below this temperature range and culture death willoccur above this temperature range. The aqueous medium is incubated fora period of time necessary to complete the biotransformation asmonitored by high performance liquid chromatography (HPLC), usually fora period of about 4 days, on a rotary shaker operating at about 220 rpm.with a throw of about 2 in. The aqueous medium is maintained at a pHbetween 6 and 8, preferably about 7, at the initiation and termination(harvest) of the fermentation process. A higher or lower pH will causethe culture to die. The desired pH may be maintained by the use of abuffer such as morpholinoethanesulfonic acid (MES),morpholino-propanesulfonic acid (MOPS), and the like, or by choice ofnutrient materials which inherently possess buffering properties, suchas production media described herein below.

The preferred sources of carbon in the nutrient medium are certaincarbohydrates such as glucose, xylose, galactose, glycerin, starch,dextrin, and the like. Other sources which may be included are maltose,rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, andthe like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other vegetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients are also suitable for use. When desired,there may be added to the medium mineral salts such as sodium or calciumcarbonate, sodium or potassium phosphate, sodium or potassium chloride,sodium or potassium iodide, magnesium salts, copper salts, cobalt salts,and the like. If necessary, especially when the culture medium foamsseriously, a defoaming agent, such as liquid paraffin, fatty oil, plantoil, mineral oil or silicone may be added.

Submerged aerobic cultural conditions are preferred for the productionof compound I in massive amounts. For the production in small amounts, ashaking or surface culture in a flask or bottle is employed.Furthermore, when the growth is carried out in large tanks, it ispreferable to use the vegetative form of the organism for inoculation inthe production tanks in order to avoid growth lag in the process ofproduction of compound I. Accordingly, it is desirable first to producea vegetative inoculum of the organism by inoculating a relatively smallquantity of culture medium with spores or mycelia of the organismproduced in a "slant" and culturing said inoculated medium, also calledthe "seed medium", and then to transfer the cultured vegetative inoculumaseptically to large tanks. The fermentation medium, in which theinoculum is produced, is substantially the same as or different from themedium utilized for the production of compound I and is generallyautoclaved to sterilize the medium prior to inoculation. Thefermentation medium is generally adjusted to a pH between 6 and 8,preferably about 7, prior to the autoclaving step by suitable additionof an acid or base, preferably in the form of a buffering solution.Temperature of the seed medium is maintained between 26° C. and 29° C.,preferably 27° C.; culture growth will be inhibited below this range andculture death will occur above this range. Incubation of the seed mediumis usually conducted for a period of about 24 to 72 hours, preferably 48hours, on a rotary shaker operating at about 220 rpm; the length ofincubation time may be varied according to fermentation conditions andscales. Agitation and aeration of the culture mixture may beaccomplished in a variety of ways. Agitation may be provided by apropeller or similar mechanical agitation equipment, by revolving orshaking the fermentor, by various pumping equipment or by the passage ofsterile air through the medium. Aeration may be effected by passingsterile air through the fermentation mixture.

The preferred culturing/production medium for carrying out thefermentation is the following medium:

    ______________________________________                                        Seed/Transformation Medium B                                                                        g/l                                                     ______________________________________                                        Glucose               10.0                                                    Hycase SF             2.0                                                     Beef Extract          1.0                                                     Corn Steep Liquor     3.0                                                     Adjust pH to 7.0                                                              ______________________________________                                    

The product, compound I, can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown biologically active substances. Compounds I is found in thecultured mycelium and filtrate, which are obtained by filtering orcentrifuging the cultured broth, and accordingly can be isolated andpurified from the mycelium and the filtrate by a conventional methodsuch as concentration under reduced pressure, lyophilization, extractionwith a conventional solvent, such as methylene chloride and the like, pHadjustment, treatment with a conventional resin (e.g. anion or cationexchange resin, non-ionic adsorption resin, etc.), treatment with aconventional adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization, andthe like. A preferred recovery method is solvent extraction,particularly using methylene chloride. A preferred purification methodinvolves the use of chromatography, especially high performance liquidchromatography (HPLC), using a silica gel column and an eluant mixturecomposed of water and an organic solvent such as methanol, acetonitrileand the like, and a small amount of acid such as acetic acid,trifluoracetic acid, phosphoric acid and the like. A preferred eluant iscomposed of water containing 0.1% trifluoroacetic acid and acetonitrileand is run through the column in a linear gradient.

The novel compound of this invention, compound I, exhibits A IIantagonist activity by the IC₅₀ assay, and therefore is useful intreating hypertension. It is also of value in the management of acuteand chronic congestive heart failure. This compound may also be expectedto be useful in the treatment of secondary hyperaldosteronism, primaryand secondary pulmonary hyperaldosteronism, primary and secondarypulmonary hypertension, renal failure such as diabetic nephropathy,glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria ofprimary renal disease, end stage renal disease, renal transplanttherapy, and the like, renal vascular hypertension, left ventriculardysfunction, diabetic retinopathy and in the management of vasculardisorders such as migraine, Raynaud's disease, luminal hyperplasia, andto minimize the atherosclerotic process. The product of this inventionis also useful for cognitive function enhancement. The application ofthe compound of this invention for these and similar disorders will beapparent to those skilled in the art.

The compound of this invention is also useful to treat elevatedintraocular pressure and to enhance retinal blood flow and can beadministered to patients in need of such treatment with typicalpharmaceutical formulations such as tablets, capsules, injectables, aswell as topical ocular formulations in the form of solutions, ointments,inserts, gels, and the like. Pharmaceutical formulations prepared totreat intraocular pressure would typically contain about 0.1 to 15% byweight, preferably 0.5% to 2% by weight, of the compound of thisinvention.

In the management of hypertension and the clinical conditions notedabove, the compound of this invention may be utilized in compositionssuch as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions orsuspensions for parenteral or intramuscular administration, and thelike. The compound of this invention can be administered to patients(animals and human) in need of such treatment in dosages that willprovide optimal pharmaceutical efficacy. Although the dose will varyfrom patient to patient depending upon the nature and severity ofdisease, the patient's weight, special diets then being followed by apatient, concurrent medication, and other factors which those skilled inthe art will recognize, the dosage range will generally be about 1 to1000 mg. per patient per day which can be administered in single ormultiple doses. Perferably, the dosage range will be about 2.5 to 250mg. per patient per day; more preferably about 2.5 to 75 mg. per patientper day.

The compound of this invention can also be administered in combinationwith other antihypertensives such as α-methyldopa, and/or diuretics suchas hydrochlorothiazide, and/or angiotensin converting enzyme inhibitorssuch as enalapril, and/or calcium channel blockers such as nifedipine.Typically, the individual daily dosages for these combinations can rangefrom about one-fifth of the minimally recommended clinical dosages tothe maximum recommended levels for the entities when they are givensingly. These dose ranges can be adjusted on a unit basis as necessaryto permit divided daily dosage and, and as noted above, the dose willvary depending on the nature and severity of the disease, weight of thepatient, special diets and other factors. Typically, these combinationscan be formulated into pharmaceutical compositions as discussed below.

About 1 to 100 mg. of compound I or a physiologically acceptable saltthereof, or any combination of these compounds or their physiologicallyacceptable salt forms, is compounded with a physiologically acceptablevehicle, carrier, excipient, binder, preservative, stabilizer, flavor,etc., in a unit dosage form as called for by accepted pharmaceuticalpractice. The amount of active substance in these compositions orpreparations is such that a suitable dosage in the range indicated isobtained.

Illustrative of the adjuvants which can be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent such as corn starch,pregelatinized starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. When the dosage unitform is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccuring vegetable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, antioxidants and the like can beincorporated as required.

RECEPTOR BINDING ASSAY USING RABBIT AORTAE MEMBRANE PREPARATION

Three frozen rabbit aortae (obtained from Pel-Freeze Biologicals) weresuspended in 5 mM Tris-0.25M Sucrose, pH 7.4 buffer (50 ml) homogenized,and then centifuged. The mixture was filtered through a cheesecloth andthe supernatant was centrifuged for 30 minutes at 20,000 rpm at 4° C.The pellet thus obtained was resuspended in 30 ml of 50 mM Tris-5 mMMgCl₂ buffer containing 0.2% Bovine Serum Albumin and 0.2 mg/mlBacitration and the suspension was used for 100 assay tubes. Samplestested for screening were done in duplicate. To the membrane preparation(0.25 ml) there was added ¹²⁵ I-Sar¹ Ile⁸ -angiotensin II [obtained fromNew England Nuclear] (10 ul; 20,000 cpm) with or without the test sampleand the mixture was incubated at 37° C. for 90 minutes. The mixture wasthen diluted with ice-cold 50 mM Tris-0.9% NaCl, pH 7.4 (4 ml) andfiltered through a glass fiber filter (GF/B Whatman 2.4" diameter). Thefilter was soaked in scintillation cocktail (10 ml) and counted forradioactivity using Packard 2660 Tricarb liquid scintillation counter.The inhibitory concentration (IC₅₀) of potential AII antagonist whichgives 50% displacement of the total specifically bound ¹²⁵ I-Sar¹ Ile⁸-angiotensin II was presented as a measure of the efficacy of suchcompounds as AII antagonists.

Using the methodology described above, the compound of this inventionwas evaluated and was found to exhibit an activity of at least IC₅₀ <50μm thereby demonstrating and confirming the utility of the compound ofthis invention as an effective AII antagonist.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Preparation of Substrate Compound II3-[(2'-tetrazol-5-yl-biphenyl-4-yl)methyl]-7-methyl-2-propyl-3H-imidazo[4,5-b]pyridine

I. Preparation ofN-triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole

Step 1: 2-cyano-4'-methylbiphenyl

To a solution of p-bromotoluene (30 g) in dry ether (150 ml) at -78° C.,a solution of t-BuLi in pentane (1.7M) (210 ml) was added slowly over aperiod of 1 hour and 30 minutes, using a dropping funnel. The bath wasthen removed and the mixture was stirred at room temperature for anadditional 2 hours. The content of the flask was then added slowly(using a cannula) at room temperature to a premixed solution of ZnCl₂ inether (1M) (180 mL) and dry tetrahydrofuran (360 mL). The mixture wasstirred for 2 hours at that temperature and the slurry was added (usinga cannula) to a solution of 2-bromobenzonitrile (21.3 g) and NiCl₂ (Ph₃P)₂ (2.1 g) in dry tetrahydrofuran (300 ml). The mixture, after stirringat room temperature overnight (18 hours), was poured slowly withstirring into ice cold 0.5N HCl (1500 ml). The organic layer wasseparated, and the aqueous phase was extracted with ether (3×300 ml).The combined organic layer was washed with water, brine and then driedover MgSO₄. Removal of the solvent gave the crude product as a semisolidmass (34 g). The material was purified on a silica gel flash columnusing ethyl acetate/hexane (1:12) to give the desired nitrile as a lowmelting solid (28 g, 88%).

¹ H NMR (CDCl₃) δ 2.42 (s, 3H), 7.2-7.8 (m, 8H); FAB-MS: m/e 194 (M+H).

Step 2: Trimethylstannyl azide

To a concentrated solution of NaN₃ (40 g) in water (100 ml), a solutionof trimethyltin chloride (20 g) in dioxane (10 ml) was added in threeportions under vigorous stirring. An instantaneous precipitate formationwas observed. The mixture, after stirring overnight at room temperature,was filtered. The residue was washed with water, and dried under suctionand then in vacuo over P₂ O₅. Yield 18.7 g (81%), mp 132°-136° C.

Step 3: N-Triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole

The titled compound was prepared starting from 2-cyano-4'-methylbiphenyl(Step 1) as described in European Patent Application EP 0,291,969.

II. Preparation of 1H-2-propyl-7-methylimidazo[4,5-b]pyridine

A mixture of butyric acid (6.57 mL, 71.9 mmol), 2,3-diamino-4-picoline(8.05 g, 65.4 mmol) (Lappin, G. R., Slezak, F. B. J. Am. Chem. Soc.(1950) 72, 7806-7) and polyphosphoric acid (50 g) was heated to 100° C.with stirring for 3 hours. The reaction was monitored by tlc of NH₄ OHneutralized aliquots. Basification (NH₄ OH), extraction(dichloromethane, 4×50 mL), drying (K₂ CO₃), purification (by filteringthrough 100 g SiO₂, ethyl acetate elution), and concentration gave 10.0g (95%) of the title compound as an amorphous tan solid which was judgedpure by ¹ H NMR.

¹ H NMR (300 MHz, CDCl₃) δ 8.13 (d, 1H, J=5 Hz), 7.01 (d, 1H, J=5 Hz),3.01 (t, 2H, J=7.8 Hz), 2.67 (s, 3H), 2.07-1.93 (m, 2H), 1.06 (t, 3H,J=7.5 Hz).

III. Preparation of3-[(2'-tetrazol-5-yl-biphenyl-4-yl)methyl]-7-methyl-2-propyl-3H-imidazo[4,5-b]pyridine

Step 1

To a stirred suspension of NaH (6.47 mmol of an 80% dispersion) in drydimethylformamide (30 mL) at room temperature was added7-methyl-2-propylimidazo[4,5-b]pyridine (991 mg, 5.66 mmol) in oneportion. After 20 minutes, the mixture was cooled to 0° C. andN-triphenylmethyl-5-(4'-bromomethylbiphenyl-2-yl)tetrazole (3.0 g, 5.39mmol) was added in one portion. The resulting mixture was warmed to roomtemperature and stirred for 15 hours. The excess NaH was quenched withwater and the bulk of the dimethylformamide was removed in vacuo at40°-50° C. The residue was extracted with ethyl acetate from brine,dried (K₂ CO₃), and concentrated. Purification by flash chromatography(SiO₂, solvent gradient: 80% ethyl acetate/hexanes, 100% ethyl acetate)gave 1.11 g (32%) thick oil of7-methyl-2-propyl-3-[2'-(N-triphenylmethyltetrazol-5-yl)biphen-4-yl]methyl-3H-imidazo[4,5-b]pyridine,Rf: 0.80 (SiO.sub. 2 tlc, 1:1 ethyl acetate: hexanes).

¹ H NMR (300 MHz, CDCl₃) δ 8.20 (d, 1H, J=5 Hz), 7.89 (d, 1H, J=8 Hz),7.51-7.39 (m, 2H), 7.38-7.20 (m, 10H), 7.10-7.03 (m, 3H), 6.95-6.88 (m,8H), 5.49 (s, 2H), 2.78-2.68 (m, 2H), 2.69 (s, 3H), 1.83-1.60 (m, 2H),0.91 (tr, 3H, J=5.5 Hz).

Step 2

To a stirred solution of the trityl-protected tetrazole (1.01 g, 1.554mmol) in dichloromethane (40 mL) at room temperature was added 85%formic acid (60 mL). After 45 minutes, the mixture was concentrated andthe residue was purified by chromatography (SiO₂, 85:13.5:1.5 CHCl₃ :CH₃ OH: NH₄ OH) followed by crystallization from 30 mL of MeOH to give583 mg, 1.429 mmol (92%) of an amorphous solid. Melting point 195°-197°C. (ethyl acetate).

¹ H NMR (300 MHz, CD₃ OD) δ 8.16 (d, 1H, J=5 Hz), 7.60-7.38 (m, 4H),7.12 (d, 1H, J=5 Hz), 7.09 (apparent singlet, 4H), 5.52 (s, 2H), 2.83(t, 2H, J=5 Hz), 2.64 (s, 3H), 1.79-1.60 (m, 2H), 0.95 (t, 3H, J=5.5Hz).

EXAMPLE 2 Microorganism and Culture Conditions

A frozen vial (2.0 ml) of culture (MA 6750) ATCC No. 55042 was used toinoculate a 250 ml baffled shake flask containing 50 ml of previouslyautoclaved (sterilized) medium A*, as noted below, used as seed medium.The pH of the seed medium was adjusted to 7.0 before autoclaving. Theseed was incubated in the seed medium at 27° C. for 24 hours on a rotaryshaker operating at 220 rpm. A 5 ml aliquot of the resulting seed mediumwas used to inoculate five 250 ml non-baffled shake flasks, eachcontaining 50 ml of previously autoclaved medium B*, used astransformation medium. Substrate compound II was added as an aqueoussolution with pH of 8 to achieve a final concentration of 0.1 mg/ml ineach flask. The resulting five transformation flasks with their contentswere subsequently incubated for 24 hours at 27° C. on a rotary shakeroperating at 220 rpm. The resultant broths were combined for isolationand purification.

ISOLATION AND PURIFICATION PROCEDURE FOR THE BROTH

The whole broth (about 250 ml) of transformation medium B was acidifiedto pH 3.5 and extracted three times with methylene chloride (3×500 ml).Methylene chloride extracts were combined, dried over sodium sulfate,and concentrated under vacuum to an oily residue. The residue wasdissolved in methanol, then subjected to high performance liquidchromatography (HPLC) purification.

HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm×25 cm columnat room temperature and monitored at 255 nm and 275 nm. The column wasdeveloped at 3 ml/min with linear gradient from 0.1% aqueous TFA:CH₃ CN,78:22 to 0.1% aqueous TFA:CH₃ CN, 40:60 in 50 minutes. The compound werecollected during repeated injections of the above described extract. Thecompound was visible at 255 nm and 275 nm. The fractions at retentiontime 16.3 minutes were pooled and the solvent then removed to yield 4.5mg of compound I.

CHARACTERIZATION

Compound I of this invention was identified via NMR spectroscopyyielding the proton NMR spectrum of FIG. 1 and the ¹³ C NMR of FIG. 2,which also contain the assigned molecular structure.

What is claimed is:
 1. A compound of structural formula I: ##STR6##or apharmaceutically acceptable salt thereof.
 2. An antihypertensivepharmaceutical composition comprising a non-toxic therapeuticallyeffective amount of the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 3. A method of treating hypertension or congestiveheart failure comprising the administration of a non-toxictherapeutically effective amount of the compound of claim 1 to a subjectin need of such treatment.
 4. A process for the preparation of acompound represented by formula I ##STR7##comprising the steps ofculturing a microorganism Streptomyces sp. (MA 6750) (ATCC No. 55042) ina nutrient medium containing assimilable sources of nitrogen and carbonand substrate compound II ##STR8## under aerobic conditions until asubstantial amount of the compound is produced and isolating thecompound so produced.
 5. The process of claim 4 wherein the temperatureis 26°-29° C.
 6. The process of claim 5 wherein the temperature is 27°C.